Type 1 interferons (IFNs) were discovered as mediators of host antiviral functions, and were recently shown to play essential roles in the innate immune response to nonviral pathogens. Established effectors of IFN antiviral action may thus serve previously unrecognized roles in antibacterial immunity. To understand how IFN exerts its antibacterial activities, the identities and mechanisms of action of specific antibacterial effectors must be determined. Towards this goal, we identified a novel role for RNase-L, an IFN-regulated antiviral ribonuclease, in the host antibacterial response. RNase-L-/- mice exhibited a significant increase in mortality following bacterial challenge. The increased susceptibility of RNase-L-/- mice to bacterial challenge was due to a compromised immune response resulting in an increased bacterial load. Microarray analysis to identify RNase- L-regulated host transcripts that may mediate its antibacterial activity identified two classes of RNase-L- dependent gene regulation that affected transcripts encoding established immune mediators. Specifically, in RNase-L-/- macrophages: 1. basal expression of cathepsin-E (CatE), that mediates endolysosome-associated immune functions, was increased suggesting that this mRNA is an RNase-L substrate; and 2. bacteria-induced expression of proinflammatory cytokines and chemokines was diminished, suggesting that RNase-L indirectly impacts their expression via the modulation of upstream signaling pathway(s). Consistent with the altered CatE expression in RNase-L-/- macrophages, the endolysosome-mediated clearance of phagocytic vacuoles was disrupted following bacterial infection. Bacteria-induced signal transduction was impaired in RNase-L-/- macrophages, providing a mechanistic basis for the diminished cytokine and chemokine expression, and corresponding modulation of neutrophil recruitment. Based on these findings, we hypothesize that RNase-L exerts its antibacterial activity through multiple mechanisms including the regulation of CatE- mediated endolysosome functions, and the induction of host cytokines and chemokines. The goals of this proposal are to dissect the mechanisms by which RNase-L regulates the expression of host immune response genes (aims 1 and 2), and to determine the roles of this regulation in its antibacterial activity (aim 3). The antibacterial activity of RNase-L activators will also be examined. The proposed studies will provide the first information on the role of RNase-L in antibacterial activity, and will identify specific components of RNase-L action as targets for therapeutic modulation of the innate immune response. Public Health Relevance: Bacterial infections are a major public health problem, and the mechanisms by which the immune system combats bacterial infection are incompletely understood. The identification of key mediators of bacterial defense is essential for the development of therapies to enhance the antibacterial immune response. The proposed studies will provide the first information on the role of RNase-L as a novel mediator of antibacterial activity, and will evaluate the antibacterial activity of RNase-L activators as a new class of therapeutic agent.